ABSTRACT Aggressive variant prostate cancer (AVPC) is a highly lethal form of prostate cancer (PCa) that arises in men who have failed treatment with second-generation anti-androgen therapy. Not driven by the androgen receptor- signaling axis, effective treatment options do not exist for AVPC and new, innovative therapies are urgently needed. Radioimmunotherapy agents targeting the cell-surface protein prostate cancer-specific membrane antigen (PSMA) have shown promise in treating patients with certain subsets of PCa. PSMA is not expressed in AVPC and there is a dire need to identify targetable antigens specific to AVPC for RIT. The overall goal of this project is to create clinically relevant radioimmunotherapy strategies for AVPC by targeting newly identified cell- surface antigens unique to the disease with novel antibody constructs and by using radiosensitizing small- molecule drugs to increase the effectiveness of RIT. Recently, we identified a novel single chain variable fragment (scFv) from a human antibody phage display library that specifically bound to a glycosylation- independent epitope on the peptide backbone of the transmembrane protein CD133. Commonly identified as a cancer stem cell marker, the function of CD133 in cancer is unknown. All commercially available antibodies for CD133 recognize glycosylation-dependent epitopes that vary between cells and at different stages of the cell cycle. Immunohistochemistry (IHC) with these antibodies yields inconsistent results and poor staining quality; thus contributing to our limited knowledge of CD133. Our scFv for CD133, termed A10, was converted into a full- length immunoglobulin (IA10) for IHC analysis on patient biopsies and tissue microarrays. Remarkably, we found that CD133 was only expressed in soft tissue and bone metastases of men who failed second-generation anti- androgen therapy and developed AVPC. As a nuclear imaging agent, IA10 was able to image CD133 expression in vivo. In biodistribution studies, IA10 and a smaller minibody construct version (MA10) both demonstrated high tumor uptake and favorable pharmacokinetics. The high tumor uptake of the A10 antibody constructs was the direct result of their rapid internalization by CD133-expressing cells making them ideal candidates for (RIT). In this proposal, we will investigate the therapeutic potential of IA10 and MA10 as RIT agents labeled with 177Lu in cell line-derived and patient-derived xenograft models of AVPC (Aim 1), evaluate the ability of the poly(ADP- ribose) polymerase inhibitor olaparib to act as a radiosensitizing agent to increase the therapeutic efficacy of RIT agents (Aim 2), and identify novel antigen-antibody combinations for RIT from AVPC tumor-derived organoid models using antibody phage display (Aim 3). Our preliminary data strongly suggest that we have developed potent RIT tools for AVPC that possess the potential to result in a dramatic shift in how the disease is treated.